Synthetic active peptide fragments

ABSTRACT

The present invention relates to peptide fragments which have one or more shared and/or similar amino acid sequences to amino acid sequences of specific portions of the 14 kDa protein of  S. mansoni  (Sm14) or related FABPs (Fatty Acid Binding Proteins), the said peptide fragments functioning as continuous or discontinuous epitopic regions of the molecule or mimicking its biological activity. More particularly, the present invention relates to a method for constructing active peptide fragments, peptide fragments, immunogenic composition and diagnostic kit using said peptide fragments.

The present application is a divisional of U.S. application Ser. No.11/005,566, filed Dec. 7, 2004, which is a divisional of U.S.application Ser. No. 10/113,946, filed Apr. 2, 2002. The entire contentsof both applications are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to peptide fragments which have one ormore shared and/or similar amino acid sequences to amino acid sequencesof specific portions of the 14 kDa protein of S. mansoni (Sm14) orrelated FABPs (Fatty Acid Binding Proteins), the said peptide fragmentsfunctioning as continuous or discontinuous epitopic regions of themolecule or mimicking its biological activity. More particularly, thepresent invention relates to a method for constructing active peptidefragments, peptide fragments, immunogenic composition and diagnostictest kit using said fragments.

BACKGROUND OF THE INVENTION

Sm14, belonging to the family of Fatty Acid Binding Proteins (FABPs), isa cross reactive antigen showing a high level of protection againstschistosomiasis and fasciolosis.

Pathogens are infectious organisms, such as bacteria, virus, protozoa,helminths, or any parasite which causes infectious diseases to the hostgenerally by expressing specific antigens which are recognized by hostimmune systems as foreign and become the target of an immunologicalresponse to eliminate the infectious pathogen.

Typically, there are specific sites on antigens, the binding epitopes orjust epitopes, which bind to a complementary portion of a cellularprotein, i.e., the receptor site. Thus, pathogen antigens often bind tocellular receptors on a host's cell as part of the process of infectionof the host by the pathogen. Similar complementarity exists between hostantibodies raised against an antigen and the antigenic determinants ofthe antigen itself. These regions of the antigenic molecule, however,may be different from those important for host cell invasion. In orderto immunize the host and reduce the effectiveness of the pathogen tomount a challenge to the host, a number of vaccination strategies havebeen devised.

Up to recently, as described in Institute of Medicine, “Vaccine supplyand innovation”, Washington, D.C.: National Academy Press (1985),several strategies have been employed to develop safe and effectivevaccines consisting of live attenuated pathogens, killed pathogens,components of pathogens, or modified toxins (toxoids).

Vaccines against several pathogenic virus, bacteria, and protozoa, suchas small pox, yellow fever, measles, diphtheria and malaria areavailable. Concerning pathogenic helminths which are parasitic worms andcause human and veterinary diseases, such as schistosomiasis andfasciolosis, at the moment, no vaccines are currently used in preventionand control programmes. These diseases are not directly transmittablefrom one person (or animal) to another and the helminth requires anintermediate host and environmental conditions to complete its complexlife cycle. There is still a great gap in the knowledge of the variablesinfluencing the dynamics of transmission of these diseases in connectionwith vaccines and vaccination protocol design. In other words, and basedon the current knowledge of epidemiological parameters which modulateand influence vaccination efficacy against these diseases, it can beasserted that neither the preferential individual levels of protectionrequired by a vaccine, nor the number of individuals to be vaccinatedand/or protected among a given population have yet been established.

Nowadays, the use of vaccines composed of pathogen components orattenuated parasites for human immunization is considered impracticaland potentially dangerous. The worry in using such complex and undefinedmixtures comes from the fact that the majority of components stimulatenon-functional immune responses and some components can even bedetrimental to vaccinated subjects, when toxic products of lipidperoxidation can be generated by immune attack against other parasiteantigens, particularly surface antigens.

These considerations have led researchers to seek alternative methodsfor effective immunization and a great deal of effort has been made topurify natural proteins from natural sources or synthetically producethem by chemical means or alternatively by using recombinant DNAtechnology.

Attempts to vaccinate model animals against schistosomiasis withhomogenates led researchers to find a saline extract (SE) whichpresented good results in conferring protection against diseases causedby Schistosoma infections in humans.

Protective immunity against schistosomes, was reported on the use of a“cocktail” of schistosome components (called SE) released early duringthe incubation of live and freshly perfused S. mansoni adult worm inphosphate buffered saline (PBS). Focusing on attempts to achieveprotection against cercarial infection by vaccination, an experimentalmodel was designed, in two different outbred animal hosts, the SW mouseand NZ rabbit, known to be fully susceptible and partially resistant toS. mansoni infection respectively.

Studies on the induced immune response in vaccinated animals aiming atthe identification of the functionally relevant SE protectivecomponents, the site and mechanism of parasite death as well as markersof protection, have been the focus of our efforts in recent years. Lessinformation on the molecular composition of SE, as well as on theidentification and isolation of its protective components has beenavailable until recently. (see: Tendler, M. and Scapin, M. (1979). “Thepresence of Schistosoma mansoni antigens in solutions used for storingadult worms”. Rev. Inst. Med. Trop. 21(6): 293-296; Tendler, M et al.(1982). “Immunogenic and protective activity of an extract ofSchistosoma mansoni”. Mem. Inst. Oswaldo Cruz. 77(3): 275-283).

The U.S. Pat. No. 4,396,600 issued on Aug. 2, 1983 in the name of LuigiMessineo & Mauro Scarpin described an extract of adult Schistosomemansoni worms obtained by incubation in 0.15M sodium chloride-sodiumphosphate buffer pH 5.8. The extract contains protein, carboxydrates,and nucleic acid and or by-products of the latter component and resolvesinto four major fractions (I-IV) by gel chromatography in G-100 andG-200 Sephadex columns. Immunodiffusion tests with rabbit anti-totalextract serum reveal three precipitation lines corresponding tofractions I and II and one with III or IV. Rabbits immunized with thistotal extract are found to be totally or partially (at least 77%)resistant to a challenge infection. The saline extract antigenicmaterial is an effective vaccine for the treatment and immunization ofschistosomiasis and other schistosome infections.

Another published study is “A 14-KDa Schistosoma mansoni Polypeptide isHomologous to a gene family of fatty Acid Binding Proteins—The Journalof Biological Chemistry—vol. 266, No. 13, Issue of May 5, pp. 8447-8454,1991; D. Moser, M. Tendler, G. Griffiths, and Mo-Quen Klinkert”. Thisstudy describes the sequencing of the gene and the demonstration of thefunctional activity of Sm-14 as a protein which binds lipids.

Thus, schistosome antigens present in SE and other related helminthantigens have been cloned, sequenced, characterized, and thecorresponding recombinant proteins prepared. Examples are: Sm14 (U.S.Pat. No. 5,730,984 granted to Fundação Oswaldo Cruz on 24 Mar. 1998;Fh-15 (Perez et al. (1992). “Fasciola hepatica: Molecular cloning.Nucleotide sequence and expression of gene encoding a polypeptidehomologous to a Schistosoma mansoni Fatty Acid-Binding Protein”. J. Exp.Parasitol. 74(4): 400-407.

However, vaccines which are based on the use of proteins belonging tothe pathogen, be they altered or not, are not always easily obtainable.Difficulties in the extraction, purification, quantitative analysis andmodification of such proteins are common problems with this type ofvaccine. Solutions exist for some such cases but these may result in anadditional onus to the protein production process which goes against thegeneral principle that a vaccine should be of relatively low cost andshould be globally accessible.

As an alternative, although not without its own deficiencies, is the useof synthetic peptides as vaccines.

There were attemps to combine epitopic portions of more than one antigento raise their immunological properties. An example of this approach isdescribed in the U.S. Pat. No. 5,219,566 granted to The John HopkinsUniversity on 15 Jun. 1993 and refers to the construction ofpolypeptides based on the identification of epitopic regions which arecommon to two S. mansoni proteins. The polypeptides have epitopes whichare shared by the 200 and 38 kDa proteins of S. mansoni and are able tobind to protein epitopes but not glycan epitopes expressed on thesurface of live schistosomula of S. mansoni. The epitope (or epitopes)on the 38 kDa protein are exposed to the surface of the schistosomulawhile the epitope on the 200 kDa protein is apparently not exposed tothe surface of schistosomula. A fusion protein having portions of anybacterial protein which is well expressed, particularly using portionsof the amino terminal end of the enzyme beta-galactosidase, is includedin the invention. It is mentioned that the particular subset of adultworm antigens was selected based on its enhanced reactivity with sera ofvaccinated as compared to chronically infected mice.

Although many antigens from helminths are available and have beenstudied in connection with their protective potential only sixSchistosoma mansoni antigens were selected by the WHO (World HealthOrganization) as vaccine candidates against diseases caused byschistosomes (see Progress Report 1975-94, Highlights 1993-94-20 Yearsof Progress, Tropical Disease Research WHO, Geneva, 1995). The selectedantigens are: GST-28 kDa (also known as Sh28-GST)—a GlutathioneS-Transferase, which is located in the schistosomula or adult wormparenchyma and in the adult worm backbone; Paramyosin-97 kDa—a muscleprotein from adult worms or schistosomula; Sm23-23 kDa—a membraneprotein from adult worms; IrV5-62 kDa—a protein which is homologous tomyosin and is present in all parasite stages; TPI-28 kDa—a TriosePhosphate Isomerase and rSm14-14 kDa—from adult worms and belonging tothe Fatty Acid-Binding Protein family.

Of these six vaccine candidates against S. mansoni initially selected bythe WHO, four have been subsequently endorsed for scale-up to GMP gradeantigen production and phase I/II clinical trials in humans. Two ofthese, Sh28-GST and Sm14 are closest to reaching this reality with GSTalready in phase II clinical trials for S. haematobium in Senegal andSm14 in the final stages of scale-up. Furthermore, Sm14 is the onlyvaccine candidate to have been shown to afford significant immuneprotection against two relevant helminthic diseases of human andveterinary importance, namely Schistosomiasis and Fascioliasis.

Sm14 is thus a unique opportunity for attacking both the second mostprevalent parasitic disease in humans—Schistosomiasis—and the mostimportant helminth infection of cattle—Fascioliasis—and thereforerepresents an attractive strategy for helminth vaccine development.

However, while some success has been achieved, these molecules are quitelarge.

A method currently under intensive investigation is the use of syntheticpeptides corresponding to segments of the proteins from the pathogenicorganism against which an immune response is directed. When thesepeptides are capable of eliciting a neutralizing immune response theyappear to be ideal immunogens. They elicit a specific response andtypically do not lead to deleterious effects on the host. However, itcan be difficult to predict which peptide fragments will be immunogenicand lead to the development of a neutralizing response. It could bedesirable to develop immunogens that elicit a response to specificneutralizing epitopes without causing responses to extraneous epitopesthat could “dilute” the specific response or lead to harmful immunecomplex formation, including autoimmune reactions.

Such a method is accomplished by the identification of specific anddiscrete portions of proteins involved in the protein-proteininteractions relevant to the immune response and the construction ofbiologically active peptides based upon the amino acid sequencesidentified.

Protein binding or protein-protein interactions can be broadly definedas an example of molecular recognition in which the surfaces of twomacromolecules (proteins) or a peptide and a protein present discretesurface interactions involving chemical and shape complementarity. Suchdiscrete interactions arise when residues of one protein (or peptide)are located spacially close to residues of another protein andattractive forces between the residues such as Van der Waals forces,salt bridges, hydrogen bonds, and hydrophobic interactions exist. Thethree-dimensional disposition of specific kinds of residues allowsattachment to occur as a consequence of a large number of theabove-mentioned weak interactions which together lead to a significantbinding energy between the different proteins.

The hypervariable loops that occur in the complementarity determiningregions of antibodies for example, on interacting with antigen epitopesmay employ the wide range of chemical interactions described above. Thebinding surface or cavity on the antibody (paratope) is formed by thespacial distribution of the residues which comprise the variable domainof the antibody's light and heavy chains and particularly thehypervariable regions responsible for antigen complimentarily. Good fitof the antigen's epitope into the antibody's paratope depends on theshape and chemical nature of both components. The affinity of a givenantibody for its antigen depends on the sum of the attractive andrepulsive forces between epitope and paratope. However, since anantibody possesses two paratopes and given that many antigens aremultivalent in nature, the overall antibody avidity will depend on thetotal number of paratopes and epitopes involved in the interaction.

A wide variety of topographies are observed for antibody combiningsites. They may be relatively flat surfaces (common in the case ofprotein antigens), grooves (as is often the case for peptides) orcavities (in the case of small molecule haptens). Often exposed,flexible and highly protruding parts of a protein antigen (oftencorresponding to surface loops on the structure), are the immunodominantepitopes and there is evidence to suggest that there is flexibility inboth the antigen and antibody which is necessary for an optimal‘induced’ fit on complex formation.

Similarly for other types of molecular recognition important in theimmune response, individual structural elements of the proteins involvedare fundamental for the specificity. This is true for example in HLAinteractions with processed peptides and in the interaction of theT-cell receptor with such HLA-peptide complexes.

By identifying the specific and discrete portions which confer antigenicproperties to a particular protein, biologically active peptides can beconstructed to mimic pathogen antigens and act on mammalian cells bybinding to the receptor sites of those cells to alter or affect theirfunction or behavior, or to prevent or alter the effect which pathogenantigens would otherwise have upon those cells. Such mimicking moleculeswould be useful as agents to affect the cells in the same manner as thenatural protein. Alternatively such peptides may bind to solubleantibody.

As such, active peptides derived in this manner may elicit eitherT-lymphocyte and/or B-lymphocyte immune responses. Accordingly, thedocument WO91/09621 discloses a peptide fragment bearing amino acidsequences of the 28 kDa Schistosoma mansoni antigen which shares atleast one epitope which induces a T-lymphocytes specific response and atleast one epitope which induces a B-lymphocytes specific response. Thepeptide fragment described in that patent application corresponds to2^(n)-fold the amino acids 115-131 of the 28 kDa Schistosoma mansoniprotein. It is mentioned that the advantage in using such a peptidefragment is to induce both humoral and cellular responses while theoriginal protein (28 kDa of S. mansoni) does not induce almost anyhumoral response. It is also mentioned that the peptide fragment confersa protection of about 40-50% in animal models (rats).

EP 251 933 proposes a process for isolating a peptide fragment bearingat least one epitope of the 28 kDa of S. mansoni by subjecting, undercontrolled proteolytic conditions, the 28 kDa polypeptide of S. mansonito the action of the protease V8. The applicant mentions that thepreferred peptide fragments are those of 8 kDa and of 6 kDa which bearthe anti-Schistosoma antigenic activity shown by the 28 kDa protein. Theamino acid sequences of the peptide fragments were not disclosed in thepatent application.

Synthetic vaccines which comprise a peptide fragment of sufficient sizeare considered to be of critical importance in providing the activeportion or portions of the entire antigen which can be recognized by theimmune system and evoke formation of the corresponding antibodies.Biologically active peptides can be constructed which function as theepitope or mimic a biologically active protein. Alternatively,biologically active peptides can be constructed which interact withreceptors and thereby block the binding of a pathogen antigen orbiologically active protein to a receptor.

U.S. Pat. No. 5,019,383 describes a synthetic vaccine comprising apeptide residue coupled to one or more alkyl or alkenyl groups of atleast 12 carbon atoms in length or other lipophylic substance. It isdescribed that the peptide residue contains a sequence of 6 amino acidscorresponding to the sequence of such amino acids in a protein antigenor allergen where the greatest local average hydrophilicity of theantigen or allergen is found. Moreover, it is mentioned that the alkylor alkenyl groups are the carrier on which the peptide residue isdisposed, said carrier being of critical importance in providing theactive portion of the synthetic peptide chain with sufficient size sothat the entire synthetic antigen or synthetic allergen can berecognized by the immune system and evoke formation of the correspondingantibodies. It is also described that the synthetic peptide residue hasa chain length of minimally six amino acids, preferably twelve aminoacids and can contain an infinitely long chain of amino acids or theircomponents, which can be characterized by the presence of other epitopesof the same or different antigen or allergen.

Another example of an active peptides approach is disclosed in thepatent application WO93/23542 which refers to nucleic acid moleculescontaining nucleotide sequences encoding helminth aminopeptidaseenzymes, and antigenic fragments and functionally-equivalent variantsthereof, their use in the preparation of vaccines for use againsthelminth parasites, and synthetic polypeptides encoded by them. Theinvention of WO93/23542 is based upon the role of mammalian integralmembrane aminopeptidases in cleaving the small peptides which are thefinal products of digestion.

In short, the identification of epitopic regions of pathogen antigens orbiologically active proteins can be used in the construction ofbiologically active compounds which comprise equivalent or shared aminoacid sequences. Furthermore, biologically active compounds, such aspeptides, can be modelled based upon amino acid sequences ofbiologically active proteins whose epitopic regions are known.

However, peptides are basically fragments of polypeptide chain oflimited size, which may be sometimes modified with respect to theoriginal sequence of amino acids as found in the parent protein fromwhich the fragment is derived. This presents a series of problems.Factors such as peptide solubility, degradation, aggregation,conformational stability, among others, are relevant given the finalobjective intended for the peptide.

As discussed above, Sm14 is a protein of particular interest. It is across reactive antigen which confers protection against bothschistosomiasis and fasciolosis (see Tendler, M. et al. (1996). “ASchistosoma mansoni fatty acid binding protein, Sm14, is the potentialbasis of a dual-purpose anti-helminth vaccine” Proc. Natl. Acad. Sci.93: 269-273 and U.S. Pat. No. 5,730,984). The data presented in thesepublications show the effectiveness of Sm14 in conferring high levels ofprotection against helminth infections.

Thus, the antigen Sm14 is an especially suitable active protein whichcan be used to model biologically active peptides based upon thedetermination of its cross-reactive epitopes.

SUMMARY OF THE INVENTION

The present invention relates to peptide fragments which have one ormore shared and/or similar amino acid sequences to amino acid sequencesof specific portions of the 14 kDa protein of S. mansoni, Sm14, orrelated FABPs, the said peptide fragments functioning as the continuousor discontinuous epitopic regions of Sm14

It is an object of the invention to prepare active peptides havingidentical or appropriately substituted amino acid sequences to those ofamino acid sequences of one or more epitopic regions of the Sm14 antigenor related FABPs.

It is another object of the present invention to provide a process forconstructing active peptides which mimic the Sm14 antigen or relatedFABPs or prevent the interaction between helminth pathogens andreceptors.

It is yet another object of the present invention to provide animmunogenic compositions able to confer at least partial protectionagainst infection with pathogenic helminths, and thus serve as vaccinesagainst same.

It is still another object of the present invention to provide adiagnostic test for helminth infection diagnostics using active peptideshaving similar or appropriately modified amino acid sequences to thosefound in epitopic portions of the Sm14 antigen or related FABPs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the sequence alignment of members of the FABP familyused for comparison of Sm14 and Fh15 with other FABPs.

FIG. 2A schematically illustrates the basic architecture of FABPs.

FIG. 2B schematically illustrates the ribbon diagram of the molecularmodel for Sm14 constructed on the basis of the three-dimensionalstructures of the homologous molecules from mouse adipocyte, ratintestine, and human muscle. The β-sheet strands in the C-terminalregion of the molecule (from residue 85 onward) are shown in a lightershade of grey. Residues shown in ball-and-stick representation areidentical in both Sm14 and Fh15 and are solvent exposed. The Figure wasproduced with the program RIBBONS (see Carson, M. (1987). J. Mol.Graphics. 5:. 103-106).

FIG. 3 shows sequence alignment of FABPs highlighting β-sheets (lightgray indicates β-sheets and dark gray indicates β-bulges) and theposition of Sm14 residues which suggest immunogenic relevance (#indicates residues which are solvent accessible, these residues beinglocated at the following positions: 1, 18, 22, 26, 41, 43, 48, 63, 66,86, 88, 89, 90, 93, 99, 106, 109, 111, 119, 120, 121 and 122; $indicates the residues that are not solvent accessible, said residuesbeing located at the following positions: 42, 49, 80, 94, 114, 125).These are identical to those indicated in FIG. 1.

FIG. 4 shows local sequence identity in pairwise comparisons betweenhuman and parasite fatty-acid binding proteins. Sequence identity iscalculated within a sliding window of 21 residues and plotted as afunction of the central residue for comparisons of Sm14 with humanadipocyte FABP (series 1), cellular retinoic acid binding protein I(series 2), cellular retinoic acid binding protein II (series 3),cellular retinol binding protein I (series 4), Fh15 (series 5),intestinal FABP (series 6) liver FABP (series 7), muscle FABP (series8), P2 myelin protein (series 9) and psoriasis-related FABP homologue(series 10). Towards the C-terminus (after alignment position 90) thecurve corresponding to the comparison between Sm14 and Fh15 is clearlydistinguishable from the remainder.

FIG. 5 illustrates schematically the formation of the fragments of thediscontinuous epitope. As can be seen two regions which participate in apredicted discontinuous epitope are chosen to form components of acontinuous synthetic peptide.

FIG. 6 provides general information about β-turns showing a graphicrepresentation (Ramachandran plot) for preferred conformations of β-turnresidues n+1 and n+2 located between β-sheets, for different turn types(I, II, I′, II′). The preferences for different amino acid types at thefour residue positions which participate in the β-turn have beenestablished in previous art (for example Wilmot C. M. & Thornton J. M.(1988) “Analysis and Prediction of the different types of β-turn inproteins” J Mol Biol 203, 221-232).

FIG. 7 is the nomenclature and composition of β-loop elements of FIG. 6.

FIG. 8 schematically represents some of the regions from Sm14 which areof immuno/antigenic interest. The regions indicated 1*-2* (whichcomprises the connection between β-strands 1 and 2 and is composed oftwo α-helices) and 9-10, belong to family 1 as defined in the text.Those indicated 6-7 and 8-9 (being the connections between thecorresponding β-strands) belong to family 2.

FIG. 9 shows the relationship between the three-dimensional structure ofSm14 and the peptides selected for vaccination trials. Specifically theregions of the molecular surface which correspond to the epitopicresidues identified by Tendler et al. (1996) are shown (top center) assticks. Center (left and right) show (in grey) how these residues maponto the molecular surface (in white). Single peptides and fusions oftwo peptides aim to represent the molecular surface corresponding to theepitopic residues as well as possible. The parts of the molecularsurface corresponding to peptides 1.1 and 2.1 are shown in grey, andthose corresponding to 1.2 and 2.2 in black (bottom left and right).

FIG. 10 shows the representation of secondary structure elements of Sm14which was the template to model the peptides of the invention. Unshadedresidues indicate connections between elements of secondary structure;($) indicates solvent-inaccessible residues of epitopes; (#) indicatesolvent-accessible residues of epitopes. The peptides of the inventioncan be ready located with reference to Tables 1 and 2.

FIG. 11 shows A first vaccination experiment against S. mansoni.Percentage protection, {(C−V)/C}×100}, of outbred Swiss mice is givenafter vaccination and subsequent challenge with 100 cercariae. From leftto right the bars correspond to vaccination with peptide 1.2, peptide1.3, peptide 1.4, peptide 2.1, peptide 2.2, peptide 2.3, peptide 2.4,peptide 2.5, peptide 2.6, r-Sm14, saline extract (SE) administered viathe footpad, SE administered via the inguinal route, adjuvant and PBSrespectively. All peptides were administered via the -inguinal route.All peptides were administered in the presence of the adjuvantmonophosphoryl lipid A+trehalose dicorynomycolate (MPL-TDM, RibiImmunoChem Research Inc.)+Alum.

FIG. 12 shows a second vaccination experiment against S. mansoni.Percentage protection is given as in FIG. 9. From left to right the barscorrespond to peptide 1.1, peptide 1.3, peptide 1.4, peptide 2.1,peptide-2.5, r-Sm14, adjuvant and PBS respectively. In this experimentall samples were administered via the footpad. All peptides wereadministered in the presence of adjuvant: monophosphoryl lipid A+trehalose dicorynomycolate (MPL-TDM, Ribi ImmunoChem ResearchInc.)+Alum.

FIG. 13 shows the percentage protection of 10 outbred Swiss mice aftervaccination and subsequent challenge with three F. hepaticametacercariae. Due to the limited number of parasites used forchallenge, animals are considered protected when they present sterileimmunity, ie no adult worms are present after sacrifice. The meanprotection is therefore given as the percentage of sterile animals atsacrifice. From left to right the bars correspond to peptide 1.1,peptide 1.3, peptide 1.4, peptide 2.1, peptide 2.5, r-Sm14 +adjuvant,r-Sm14, adjuvant and PBS respectively. The adjuvant used wasmonophosphoryl lipid A +trehalose dicorynomycolate (MPL-TDM, RibiImmunoChem Research Inc.)+Alum.

FIG. 14 illustrates parasite FABPs. Alignment showing secondarystructure elements of Sm14 and its residues which have a relevant rolein eliciting immunogenic response (# indicates solvent accessibleresidues and $ indicates the residues that are solvent inaccessible).

FIG. 15 schematically represents the position of Cysteine residues inpeptides and their role in restraining conformational movement (dashesindicate Cα).

FIG. 16A shows Western Blotting data related to extracts from differenthelminths.

FIG. 16B also shows Western Blotting data related to extracts fromdifferent helminths.

DETAILED DESCRIPTION OF THE INVENTION

For convenience, the meaning of certain terms and phrases employed inthe specification, examples, and appended claims are provided below.

The term “active protein” refers to proteins which bind to cellularreceptors and thereby alter or affect the function or behavior of thecells, or prevent or alter the effect which another biologically activeprotein would otherwise have upon those cells. A pathogen antigen can bea biologically active protein if, upon binding to a host cell, it altersor affects the function or activity of a cell or prevents another agentfrom doing so.

As used herein the term “neutralizing epitope” refers to the portion ofa pathogen antigen against which antibodies have a neutralizingactivity. That is, antibodies specific for a neutralizing epitope renderthe pathogen non-infective and/or inactive.

The term “receptor site” refers to the portion of the receptor thatinteracts with a protein that binds to the receptor.

The term “active peptides” refer to proteinaceous molecules which mimicbiologically active proteins or prevent the interaction betweenbiologically active proteins and receptors, where receptors may bemolecules of the immune system including antibodies.

The terms “correspond” and “corresponding” refer to the level of sharedidentity between two amino acid sequences and the terms “homologous”,“homology”, and “sequence similarity” are often used interchangeably bythose having ordinary skill in the art to refer to related amino acidsequences.

It has been amply discussed here that the state of the art teaches thatit has been verified that the Sm14 protein offers protection in animalmodels (Swiss mice and New Zealand rabbits) which have been infectedwith Schistosoma mansoni and previously stimulated with Sm14.Furthermore, parallel experiments in which animal models were infectedwith Fasciola hepatica, causative agents of Fasciolose, after previouslybeing stimulated with Sm14, also demonstrated the existence of aprotective cross reactivity.

Sm14 belongs to the intracellular Fatty Acid-Binding Protein family—theFABPs, whose amino acid sequence is shown in FIG. 1 and whosethree-dimensional structure can be schematically represented as shown inFIG. 2A.

Fh15, from Fasciola hepatica also belongs to the intracellular FattyAcid-Binding Protein family. Its amino acid sequence is also given inFIG. 1 and it is also schematically represented in FIG. 2A.

FIG. 1 also shows the amino acid sequences of a series of host (human)FABPs. From the figure it can be verified that Sm14 and Fh15 show adegree of sequence identity which is of a similar order to that observedbetween Sm14 and many of the remaining (host) FABPs. This is of theorder of 35 to 40%.

Specifically, in FIG. 1 the first nine sequences are all of proteinsderived from human tissues: myelin P2 from peripheral nerve; adipocyteFABP, aFABP; cellular retinol-binding protein I, cRBPI; cellularretinoic acid-binding proteins I and II, cRABP1 and cRABP2;psoriasis-related FABP homologue, pFABP-hom; intestinal FABP, iFABP;liver FABP, 1FABP; and heart FABP, hFABP. FABPs from mouse adipocyte andrat intestine are also shown, as they were used together with hFABP forthe construction of molecular models for Sm14 and Fh15 (Tendler, M. etal. (1996). “A Schistosoma mansoni fatty acid binding protein, Sm14, isthe potential basis of a dual-purpose anti-helminth vaccine”. Proc.Natl. Acad. Sci. 93: 269-273). The β-sheet strands of a FABP areindicated by the hatched blocks and numbered consecutively; the twoα-helices (h1 and h2) are marked by the solid bars. Identical residuesin the two parasite sequences (Sm14 and Fh15) are boxed. The subset ofthese residues which are conserved in no more than three of the humansequences are indicated either by stars (for exposed residues) or by thepercent symbol (for solvent inaccessible residues).

As can be seen from FIG. 1 the alignment of the host and parasite (Sm14and Fh15) FABPs together with two sequences of known three-dimensionalstructure (aFABP and iFABP) permits the definition of regions which arestructurally equivalent. These were used in the construction of themodel of Sm14 and Fh15 described in Tendler, M. et al. (1996). “ASchistosoma mansoni fatty acid binding protein, Sm14, is the potentialbasis of a dual-purpose anti-helminth vaccine”. Proc. Natl. Acad. Sci.93: 269-273 and have also been used in the construction of models forFABPs from Schistosoma japonicum (Sj14), Fasciola gigantica (Fg15) eEchinoccocus granulosus (Eg15) based on the alignment shown in FIG. 3.

Sm14 is as closely related to several human proteins, including P2myelin protein (≅42% sequence identity) and FABP from cardiac muscle((≅42%), as it is to Fh15 (≅44%), which is a FABP from Fasciolahepatica. However there is good evidence for immune cross-reactivitybetween the two parasite proteins, whilst there are no reports of S.mansoni patients developing auto-immune reactions. It is of interesttherefore to determine the specific characteristics of these twoparasite FABPs, in terms of regions of amino acid sequence andstructure, which are responsible for such cross-reactivity.

Despite this observation the Sm14 and Fh15 molecules do not exhibitsimple, coordinated alterations in size or continuous sequence whencompared with their human homologues. However Sm14 does show a markedfalloff in conservation with human sequences towards the C terminus(from about residue 85 onward) whereas the two parasite sequences showapproximately 47% mean identity within the same region. In general thisis the most poorly conserved region of the molecule across the family ofFABPs as a whole (Jones et al. (1988) EMBO J. 7, 1597-1604; SacchettiniJ. C. et al. (1988) J. Biol. Chem. 263, 5815-5819; Muller-Fahrow, A. etal. (1991) Eur. J. Biochem. 199, 271-276)). There is thus a notabledifference between the parasite proteins and their human homologues inthat the C-terminal region of the former shows an unusual level ofsequence conservation. This is shown in FIG. 4 where the average meanconservation within a 21 residue sliding window is calculated as afunction of sequence position for pairwise comparisons which involveSm14 and one other FABP. Only in the case of the comparison between Sm14and Fh15 is a clear peak in the graph seen towards the C-terminus. Theβ-strands of this region (from residue 85 onwards) are shown in alighter shade of grey in FIG. 2B in order to distinguish them from theremainder of the molecule. Indeed the cross-reactivity of these twoproteins has already been demonstrated experimentally (see Perez et al(1992) and Tendler et al (1996).

We (Tendler, M. et al. (1996). “A Schistosoma mansoni fatty acid bindingprotein, Sm14, is the potential basis of a dual-purpose anti-helminthvaccine”. Proc. Natl. Acad. Sci. 93: 269-273) have described molecularmodels constructed for both Sm14 and Fh15 and shown that the twomolecules adopt the same three-dimensional topology as other members ofthe FABP family. The basic architecture of FABPs is represented in theFIG. 2A.

As showed in FIG. 2B, the molecular model of Sm14 consists of a10-stranded antiparallel β-barrel with short interstrand connectionswhich generally form reverse turns (β-turns). Strands 7-10 of the barrelwith their connections loops essentially constitute the C-terminalportion of the molecule. When residues conserved (i.e. identical) inSm14 and Fh15 but present in no more than three of nine human sequenceswere plotted on the modeled 3D structure, two probable epitopes wereidentified and selected for purpose of the present invention. These arediscontinuous as they are constituted by residues which are spaciallyclose in the three-dimensional structure but distant in the amino acidsequence. Twenty-two of these residues were exposed on the surface andthus potentially contribute to B-cell mediated antigenicity. Of these 22residues, 13 were derived from the C-terminal portion of the protein,which we show to present an unusually high degree of conservation. Theexternal invariant residues are not randomly distributed about thesurface of the Sm14 molecule but rather are clustered at the upper andlower ends of the barrel (FIG. 2B) and potentially constitute functionaldiscontinuous epitopes which present significant variation from humanproteins. The clustering of 13 of the 22 conserved exposed residueswithin the C-terminal region, together with the evidence from FIG. 4indicates the importance of the C-terminal region (together withresidues from other parts of the molecule which come together to formdiscontinuous epitopes), for antigenicity. Indeed, the four interstrandconnections which are included within the C-terminal part of thestructure show pronounced peaks in the main-chain accessibility, aphenomenon often correlated with antigenic determinants.

In the current invention we describe a method for the unification of twodistinct peptides derived from the Sm14 molecule, which are distant inprimary structure (amino acid sequence) but spatially close in tertiarystructure. Said peptides belong to the same predicted discontinuousepitope. Besides simply unifying the peptides into a single largerpeptide, the current invention also describes a method for modifying theamino acid sequence of said peptides such that the resulting peptideadopts a three-dimensional structure which is similar to thecorresponding regions of the parent (Sm14) molecule or that such astructure may be energetically accessible to the peptide in solution orthat such a structure may be acquired by the peptide on complexformation with molecules of the immune system, such as antibodies. Thisapproach is applicable to other protein antigens which presentdiscontinuous epitopes.

According to the present invention the method for constructing peptideson the basis of the three-dimensional structures of homologous moleculeswherein said peptides are able to mimic or prevent the interactionbetween helminth pathogens and receptors, said method consisting of:

(i) selecting regions of the parent protein which contain a high spatialdensity of residues which form continuous or discontinuous epitopes;

(ii) give priority to maintaining the previously predicted epitopicresidues, responsible for stimulating the desired immuno/antigenicresponse, within the selected peptide sequence;

(iii) elaborate sequences which, whilst maintaining a high percentage ofpreviously identified epitopic residues, are of limited size varyingfrom 8 to 28 residues;

(iv) two peptides may be chosen, which may correspond to regions whichare distant in the primary structure (amino acid sequence) but spatiallyclose in the tertiary (three-dimensional) structure, the said regionsare chosen on the basis that the three-dimensional structure indicatesthat they can be readily united assuming that they retain their originalstructures.

(v) modifications may be made to the peptide, be it derived from asingle continuous stretch of amino acids or be it derived in the mannerdescribed in (iv), so as to favour the three-dimensional conformation asseen in the original protein, such modifications may includesubstitutions of amino acids as well as insertions or deletions of aminoacids;

(vi) residues, such as M-cystines, may be used in order to restrict theconformational freedom of the final peptide.

In step (iv) a minimal requirement is that the C-terminus of one peptideshould be spatially close to the N-terminus of the second peptide, butthere is no need to necessarily preserve the order of the peptides asexists in the original protein.

Amino acid sequence determination can be readily accomplished by thosehaving ordinary skill in the art using well known techniques. Generally,DNA sequencing of relevant genetic material can be performed and theamino acid sequence can be inferred from that information. Sequencing ofgenetic material, including cDNA, can be performed by routine methods bythose having ordinary skill in the art and thus can readily determinewhether or not one amino acid sequence corresponds to another. Thedetermination of whether sequences are corresponding may be based on acomparison of amino acid or nucleic acid sequence, and/or proteinstructure, between the proteins of interest. In the case relevant to thecurrent invention, the determination of the amino acid sequence of thepathogen antigen (Sm14) shows it to correspond to (be homologous to)members of a family of Fatty Acid-Binding Proteins (FABPs).

By determining the number of identical and conservatively substitutedamino acid residues shared between two molecules once aligned bystandard techniques, and knowing the length of the alignment, one havingordinary skill in the art can determine whether or not two sequencescorrespond. Depending on the sequence length, the two sequencescorrespond (are homologous) if they share approximately at least 80%identical and conservatively substituted amino acids of which at leastabout 28% are identical amino acids and between about 30-42%conservative substitutions.

The peptide is synthesized and mimics the pathogen or biologicallyactive protein. The peptide is formulated as a pharmaceuticalcomposition which is administered, for example, as a therapeutic toelicit the activity that the native proteins have on cells.

The following examples are illustrative of the invention and representpreferred embodiments. Those skilled in the art may know of, or may beable to find using no more than routine experimentation, otherappropriate materials and techniques such as the above mentioned aminoacid sequences and production methods.

Example 1 Method for Obtaining the Peptide

The three-dimensional structure of Sm14, as built by comparativehomology modeling and described in Tendler et al. (1996), was used asthe basis for obtaining peptide fragments for synthesis and subsequentvaccination trials. It should be noted that in previous studies wedescribed likely discontinuous epitopes responsible for the immunecross-reactivity between Sm14 and Fh15 and a summary of these resultshas been given above.

The residues predicted to participate in such epitopes were identifiedon the basis of the fact that they are identical in the two parasitemolecules and yet only poorly conserved in human homologues. Due to thefact that few of the predicted epitopic residues were present incontinuous stretches of the amino acid sequence, a design strategy waselaborated in order to incorporate more than one continuous segment intoa single unified peptide.

In order to aid in obtaining segments of the polypeptide chain whichwere of greatest interest for incorporation into peptides forvaccination purposes, the local sequence conservation of parasite andhost fatty acid binding proteins was first evaluated. This was done bycalculating the local percentage sequence identity between any twosequences within a 21 residue-sliding window. Comparisons were madebetween Sm14 and Fh15 and between Sm14 and nine human fatty acid bindingprotein homologues.

FIG. 4 shows the results of the sequence comparisons made between ninehuman fatty acid binding proteins and Sm14. On calculation of sequenceidentity within a sliding window of 21 residues, it can be readily seenthat in general the local similarity between Sm14 and human homologuesfalls off rapidly towards the C-terminus. This lack of sequenceconservation in the C-terminal part of the molecule has been commentedpreviously and is in direct contrast to the pattern observed whencomparing the two parasite FABPs, Sm14 and Fh15, in which the C-terminalthird of the molecule shows the greatest overall sequence conservation.Distinct patterns of residue conservation are thus apparent whencomparing either the parasite derived Sm14 with its host's homologues oralternatively with the cross-reacting homologue from a related parasite.This is despite the fact that the overall percentage identity along theentire sequence may be very similar in both cases (42% on comparing Sm14with human cardiac FABP or P2 myelin protein and 44% with Fh15).

This result suggests that judiciously chosen peptides carrying theepitopic residues predicted previously (Tendler, M. et al. (1996). “ASchistosoma mansoni fatty acid binding protein, Sm14, is the potentialbasis of a dual-purpose anti-helminth vaccine”. Proc. Natl. Acad. Sci.93: 269-273) and principally derived from the C-terminal region of themolecule, may be sufficiently distinct from human homologues to diminishthe risk of undesirable cross-reactivity. At the same time, by choosingthe peptides such that a reasonable number of residues conserved in Sm14and Fh15 are included, it is desired to increase chances of thesuccessful induction of a protective immune response against bothparasites.

This is clearly an important factor when considering the use of suchpeptides as multivalent anti-helminthes vaccines.

On the basis of the above sequential analysis together with thepredicted three-dimensional structure, two regions of the molecule wereselected for peptide obtention with emphasis placed on the C-terminalthird of the molecule. The first region is composed of two segments; anα-helix (located in the large connection between β-strands 1 and 2)together with the connection between β-strands 9 and 10. The peptidesderived from this region of the molecule will be henceforth referred toas family number 1. The second region comes from the opposite side ofthe β-barrel which forms the basic structure of the molecule and is alsodiscontinuous, in this case consisting of two β-hairpins. The firsthairpin is composed of β-strands 6 and 7 and the second, β-strands 8 and9, in both cases together with their connecting loops (type I and typeII′ β-turns respectively). The peptides derived from this region will bereferred to as family 2.

For each family, the two individual segments chosen were selected on thebasis of their spatial proximity in the three-dimensional structure andnot on their proximity in terms of amino acid sequence. Furthermore inchoosing the exact length of each component segment several additionalcriteria were taken into account. These included 1) an attempt toinclude a maximal number of the residues predicted in our previousstudies to form part of the discontinuous epitopes, 2) an attempt tomaintain the peptide as small as possible, 3) so as to facilitate thejoining of the two segments into a single peptide with reference to thethree-dimensional structure of the intact protein and 4) givingpreference to segments which make a significant number of internalcontacts.

The third and fourth criteria were introduced in order that the twosegments of a given family might be joined into a single peptide in sucha way as to retain the possibility of maintaining the original structureas observed in the whole molecule and as illustrated in FIG. 5. This isby no means meant to imply that the peptide would naturally adopt such astructure in solution. Indeed this seems unlikely given the small sizeof the resulting peptides and the known conformational flexibility ofsuch molecules in solution. Rather it is an attempt to ensure that sucha conformation is energetically accessible, for example via induced fiton complex formation with antibody.

The resulting fusion peptide, composed of the two component segments ofa given family, was subsequently modified in an attempt to favor thenative structure. Modifications were made based on the known frequenciesof occurrence of amino acid residues in β-turns and using atomic contactquality analysis as implemented in the graphics program WHATIF (Vriend,G. (1990) J. Mol. Graph. 8, 52-56; Sibanda et al. (1985) Nature 316,170-174). In the latter case, residues which became exposed to anunfavorable chemical environment as a result of being removed from thecontext of the entire structure were substituted, such that the peptideis no longer identical in amino acid sequence to that observed in Sm14itself, but continues to correspond to it. Furthermore, this approachdoes not lead to the conclusion that such substitutions must beconservative in terms of the chemical nature of the amino acidsinvolved. Indeed given that hydrophobic residues which are buried in thenative structure will often be expected to become exposed in thepeptide, it will often be necessary to make non-conservativesubstitutions and even deletions or insertions. Some informationconcerning β-turn types is given in FIGS. 6 and 7.

In the case of family 1, this resulted in four peptides. Peptide 1.1 wasderived from the β-hairpin composed of strands 9 and 10, from residue118 to 125. Peptide 1.2 was derived from the first α-helix of thestructure, from residue 15 to 24. Peptide 1.3 was a direct fusion of 1.1and 1.2, composed of 18 residues and 1.4 was derived from 1.3 by thesubstitution of four amino acid residues, based in the criteriadescribed above.

Specifically, 1.4 has been modified in order to introduce two glycineresidues at its centre aimed at favoring a bend in the peptidemainchain. Modeling studies indicated that glycines at this positionassuming the conformation of a type I′ turn would in principal be ableto unite the two fragments whilst retaining their original structure asseen in the whole protein. Two further substitutions were made tohydrophobic residues of 1.3; the Phe at position 10 was replaced by aSer and Leu at position 17 by Val. These residues are normally hiddenwithin the hydrophobic core of the whole molecule and their substitutionby less hydrophobic residues was guided by the WHATIF atomic contactquality option. After transforming 1.3 into 1.4, the quality score rosefrom 0.23 to 0.54 suggesting the substitutions to be reasonable,assuming the conformation expected for these regions in the Sm14 itself.This case exemplifies how the use of the three-dimensional structure ofSm14 for peptide modification, which is part of the current invention,is used in practice. It involves the use of substitution of the finalresidue of the first peptide (1.1) and the first residue of the second(1.2) by glycines in order to induce a reverse turn and alsonon-conservative substitutions.

In the case of family 2, six peptides were designed using a similarstrategy. Peptide 2.1 (residues 85 to 94) came from the β-hairpinbetween strands 6 and 7. Likewise peptide 2.2 was derived from thehairpin between strands 8 and 9. Peptide 2.3 is a simple fusion of 2.1and 2.2, whilst peptides 2.4 and 2.5 are alternative modificationsthereof. In peptide 2.4 asparagine at position 3 of peptide 2.3 issubstituted by phenylalanine, glutamine at position 10 by serine, aninsertion of four residues (Asp-Pro-Thr-Gly) is made between Gln10 andIle11, and residue Asp18 in peptide 2.3 is substituted by Ala22 in 2.4.In peptide 2.5 a smaller insertion of two residues is made betweenpositions 10 and 11 of 2.3 together with the substitutions indicated inTable 2. In both cases the insertion of residues between the twofragments corresponding to 2.1 and 2.2 was made with the intention ofuniting the fragments with β-turns. In the case of 2.4 the turn typeintended was type I and in the case of 2.5, type I′.

According to the present invention alternative modifications in theoriginal fragments are carried out when it is desired, for example inorder to introduce conformational stability for the peptide.

Peptide 2.6 is identical in amino acid composition to but its sequencehas been randomized and was used as a control in immunization assays inorder to evaluate the non-specific effect of a peptide of unrelatedamino acid sequence but identical amino acid content.

All of the final peptide sequences are given in Tables 1 and 2 and thefour peptides which were directly derived from Sm14 (1.1, 1.2, 2.1 and2.2) are shown schematically in FIG. 8 and mapped onto its modelstructure in Figure 9. There sequences with respect to the original Sm14amino acid sequence can be localized by referring to the followingtables together with FIG. 10.

TABLE 1 Amino acid sequences of the peptides used in the immunizationassays. Family 1 Peptide Sequence Comments 1.1 VTVGDVTA Loop betweenβ-strands 9 (SEQ ID NO: 1) and 10 1.2 NFDAVMSKLG First α-helix (between(SEQ ID NO: 2) β-strands 1 and 2) 1.3 VTVGDVTANFDAVMSKLG Union of 1.1and 1.2 (SEQ ID NO: 3) 1.4 VTVGDVTGGSDAVMSKLG 1.3 modified (SEQ ID NO:4)

TABLE 2 Amino acid sequences of the peptides used in the immunizationassays. Family 2 Peptide Sequence Comments 2.1 EKNSESKLTQ Loop between(SEQ ID NO: 5) β-strands 6 and 7 2.2 IVREVDGDTMKTT Loop between (SEQ IDNO: 6) β-strands 8 and 9 2.3 EKNSESKLTQIVREVDGDTMKTT Union of 2.1 (SEQID NO:7) and 2.2 2.4 EKFSESKLTSDPTGTVREVDGATMKTT 2.3 modified (SEQ IDNO:8) 2.5 EKFSESKLTFDGIVREVDGATMKTT Alternate (SEQ ID NO:9) modificationto 2.3 2.6 KIGTSVFGTRTSKFDATEMVLDKEE 2.5 randomized (SEQ ID NO: 10)

FIG. 9 represents the relationship between the three-dimensionalstructure of Sm14 and the peptides selected for vaccination trialsaccording to Example 1. Top center is shown a ribbon representation ofthe model for the Sm14 molecule. Residues conserved in Sm14 and Fh15(but infrequent in human homologues)and also solvent exposed, arehighlighted in stick representation. On the left (above) is indicatedthe contribution of these residues (from family 1) to the accessiblesurface of the left-hand side of the molecule, and below, how peptides1.1 and 1.2 attempt to reproduce this surface. On the right, ananalogous representation is given for family 2.

Example 2 Peptide Synthesis

The peptides according to the present invention were synthesized byusual procedures (from the state of the art) and provided in the form ofC-terminal amides as free peptides, at a purity of greater than 97%.

Example 3 Expression of Recombinant Sm14 (r-Sm14)

In order to provide control experiments the recombinant Sm14 proteinexpressed by the pRSETA-6× His-Sm14 construct was obtained aftertransformation of chemically competent E. coli BL21(DE3) as described inRamos C. R. R et al., Mem Inst. Oswaldo Cruz, Rio de Janeiro, Vol. 96,Suppl.: 131-135, 2001, herein incorporated by reference.

Materials and Methods

The pRSET A,B,C expression system was purchased from Invitrogen. ThepET3-His (Chem & Tsonwin 1994) was obtained from the National Instituteof Genetic, Japan. All the reagents used here were of analytical grade.

Expression and Purification of recombinant Sm14

The recombinant Sm14 derived from pGEMEX expression system (Promega) waspurified as described (“A Schistosoma mansoni fatty acid bindingprotein, Sm14, is the potential basis of a dual-purpose anti-helminthvaccine”. Proc. Natl. Acad. Sci. 93: 269-273 and U.S. Pat. No.5,730,984).

The recombinant Sm14 proteins expressed by pRSETA-Sm14, pET3-His-Sm14and pRSETA-6× his-Sm14 constructs were obtained after transformation ofchemically competent E. coli BL21(DE3). The transformed clones weregrown in liquid LB (Luria Bertani medium) at 37° C. with agitation (200rpm) until a 0.6 optical density was reached at 600 nm. At this point,IPTG was added to a final concentration of 0.5 mM. The cultures weregrown for an additional 3 h in the same conditions described and thecells were harvested by centrifugation at 2,000 g. The Sm14 wasexpressed in inclusion bodies in all cases. The cells resuspensed in 50mM Tris-HCl ph 8.0, 100 mM NaCl, 10 mM EDTA, 10 MM2-mercaptoethanol weredisrupted by french pressure and the insoluble Sm14 was recovered bycentrifugation. The inclusion bodies were washed by centrifugation withthe previous solution also containing 2 M urea and finally dissolved in8 M urea at room temperature for 2 h in the same buffer. The clarifiedsupernantants were diluted 200 times by dropping in refolding solution(50 mM Tris-HCl pH8.0, 500 ml NACl, 5 mM imidazol) by stirring at roomtemperature for 18-24 h. The total volume was clarified bycentrifugation and loaded onto a C10 column (Amersham Pharmacia)containing 5 ml of Ni⁺²-charged resin (Amersham Pharmacia) previouslyequilibrated with the refolding buffer at 1 ml/min. The column waswashed with 10-20 volumes of refolding buffer containing 20 mM imidazoland the adsorbed protein was eluted by 1M imidazol in the refoldingbuffer. Fractions of 1 ml were collected. Characterization of thefractions was done by SDS-PAGE and Western-Blot according to describedprotocols (Harlow & Lane 1988, Ausubel et al. 1989, Sambrook et al.1989).

Example 4 Immunization Experiments against S. mansoni

In this experiment, outbreed Swiss mice were immunized with twointradermal/subcutaneous doses at an interval of 7 days followed by abooster injection, 21 days later. In the case of the peptides, a dose of70 μg in the presence of the adjuvant monophosphoryl lipid A+trehalosedimycolate (MPL-TDM, Ribi ImmunoChem Research Inc.) and Al(OH)₃ was usedfor all injections. The peptides used in accordance with the presentinvention were those prepared as discussed in Example 1 with theexception of peptide 1.1.

For control experiments with r-Sm14 (prepared according to Example 3)and Saline Extract (SE) (as described in U.S. Pat. No. 4,396,600) thedoses used were 10μg and 300 μg respectively. In the case of SE, tworoutes of administration were employed, via the inguinal region and thefootpad. For all other antigens, only the inguinal route was used.

For assays of protection against S. mansoni, the animals were challengedsubcutaneously with 100 cercariae, 60 days after the last immunizationand perfused 45 days later. Overall protection was calculated by theformula {(C−V)/C}×100, where C is the average number of worms in controlanimals and V is the average number of worms in vaccinated animals.

The results of this first vaccination experiment against S. mansoni inSwiss mice (FIG. 11) established an apparently significant increase inprotection after administering saline extract (one of the standardcontrols used), via the footpad as compared with the inguinal route.During this initial experiment the peptides were also administeredinguinally. As a consequence a second protocol was established for themost promising peptides but employing the footpad as the administrativeroute of choice.

FIG. 11 clearly shows a large degree of discrimination and selectivityamongst the nine peptides used in the first experiment, the levels ofprotection varying greatly from a maximum of slightly over 40% (peptides1.4 and 2.1) down to no protection at all (2.5 and 2.6). This implies aspecific immune response to the different amino acid sequences and not ageneric reaction to immunization by any foreign peptide. This isemphasized by the fact that peptide 2.6, which is unrelated to Sm14 (asit was generated by sequence randomization of 2.5) did not lead toprotection.

FIG. 11 also shows that the peptides from the first family, whichoffered the greatest levels of protection, were 1.3 and 1.4, both ofwhich correspond to different variations of fusions of the smallerpeptides 1.1 and 1.2. In a second experiment (see following example),peptide 1.1, which had been excluded from the initial trial, wassubsequently shown to be as effective as the fusion peptides, 1.3 and1.4 (see FIG. 12). Of the second family, the smallest peptide 2.1, oftotal length 10 residues offered the greatest levels of protection(42.1%). The remaining members of the second family producedprogressively lower levels of protection (FIG. 11) falling to zero forpeptides 2.5 and 2.6.

Example 5 Further Immunization Experiments against S. mansoni

In this experiment an identical protocol as Example 4 was employed for asub-group of peptides including those which appeared most promising(1.1; 1.3; 1.4 and 2.1) together with 2.5 as a control, but employingthe footpad as the route of administration.

Animals were challenged subcutaneously with 100 cercariae, 60 days afterthe last immunization and perfused 45 days later. Overall protection wascalculated by the formula {(C−V)/C}×100, where C is the average numberof worms in control animals and V is the average number of worms invaccinated animals.

In this second experiment (FIG. 12), in which only a limited number ofpeptides were tested, the performance of the most protective peptides(1.1, 1.3, 1.4 and 2.1) was equivalent to that seen for the recombinantwhole protein (r-Sm14) and comparable to that reported previously(Tendler, M. et al. (1996). “A Schistosoma mansoni fatty acid bindingprotein, Sm14, is the potential basis of a dual-purpose anti-helminthvaccine”. Proc. Natl. Acad. Sci. 93: 269-273.) showing that it may bepossible to reproduce the protective immune response generated againstSm14, with much smaller molecules derived from it and which correspondto as little as less than 10% of its total molecular weight. Peptide1.1, consisting of eight residues is the smallest of all the peptidestested and yet gave rise to protective levels of close to 50%. In thesetrials against S. mansoni relatively little appears to be gained byincreasing the size of this peptide, since 1.3 and 1.4 produced verysimilar results in terms of protection.

This implies that most of the immunogenic capacity of these largerfusion peptides is due to their N-terminal sequences, which is identicalto 1.1 and which corresponds to residues 118 to 125 of the originalSm14. Peptide 1.1 is thus derived from the C-terminal region of themolecule and its important immunogenic role is consistent with theobservation made above concerning residue conservation in this region ofthe molecule.

That the C-terminal third of the molecule contributes to the mostimportant epitopes on Sm14 is further supported by the fact that peptide2.1 (corresponding to residues 85 to 94) also affords high levels ofprotection (42.1% in the first experiment and 50% in the second).However, in this case there is a marked difference when compared withthe first family. On generating the larger fusion peptides, by addingthe 2.2 sequence to 2.1 in various different formats, the resultingpeptides are less protective. One potential explanation for this may bethat humoral responses to the peptides, which should be conformationspecific, may be ineffective if the peptides assume structures whichdisguise, occlude or alter the structure of the epitope.

At present little is known about the nature of the immune responseinduced by Sm14 although it is believed to include both humoral andcellular contributions. The cellular component of this response has beencorrelated with resistance, susceptibility and delayed typehypersensitivity (DTH)-mediated pathology. It is also known that IL-10is a key molecule in the regulation of T cell response inschistosomiasis. A population study performed by Brito et al. (Brito CF, Caldas I R, Coura Filho P, Correa-Oliveira R, Oliveira S C., “CD4+ Tcells of schistosomiasis naturally resistant individuals living in anendemic area produce interferon-gamma and tumour necrosis factor-alphain response to the recombinant 14 KDA Schistosoma mansoni fattyacid-binding protein.”, Scand J Immunol. June 2000; 51(6) :595-601.PMID: 10849370 [PubMed—indexed for MEDLINE])in a Brazilianschistosomiasis endemic area showed that the highest levels ofproliferative response to Sm14 was observed mainly in peripheral bloodmononuclear cells (PBMC) from uninfected endemic normal individuals.This suggests that T cell activity against the Sm14 antigen should bethe same mechanism associated with natural resistance against infection.

On the other hand humoral responses in schistosomiasis have beenassociated with various effector or regulatory mechanisms. IgG and IgEare directly involved in the in vitro killing of schistosome larvae inassociation with macrophages and platelets. According to Brito et al(Brito C F, Fonseca C T, Goes A M, Azevedo V, Simpson A J, Oliveira S C.“Human IgG1 and IgG3 recognition of Schistosoma mansoni 14 kDa fattyacid-binding recombinant protein.”, Parasite Immunol. January 2000;22(1):41-8.) the prevalent types of antibody against Sm14 in sera ofdifferent clinical forms of schistosomiasis are IgG1 and IgG3. They alsosuggest that effector function induced by these immunoglobulin moleculesmight be a critical component of the immune system involved inprotection induced by Sm14.

Example 6 Immunization Experiments against F. hepatica

This example refers to the use of some of the peptides described inExample 1 for vaccination against Fasciola hepatica in the Swiss mousemodel. For the Fasciola hepatica protection assay each group (10 outbredSwiss mice/group) received three intradermal/subcutaneous doses via thefootpad using an identical protocol to that described above for S.mansoni. Animals were vaccinated with either 70 μg of one of thesub-group of peptides described above (1.1, 1.3, 1.4, 2.1 and 2.5),emulsified in Ribi adjuvant (MPL-TDM) and Al(OH)₃ or 10 μg of r-Sm14 (inthe presence or absence of adjuvant), or an equivalent amount ofadjuvant alone. Vaccinated and non-vaccinated control groups weresimultaneously challenged orally with 3 metacercariae of F. hepatica 60days after immunization and the sacrifice of all animals performed 30days after challenge.

FIG. 13 shows the results of vaccination trials in Swiss mice afterchallenge with three F. hepatica metacercriae.

As anticipated from previously published data (Tendler, M. et al.(1996). “A Schistosoma mansoni fatty acid binding protein, Sm14, is thepotential basis of a dual-purpose anti-helminth vaccine”. Proc. Natl.Acad. Sci. 93: 269-273), r-Sm14 in the presence or absence of adjuvantis able to offer 100% protection to outbred Swiss mice under the givenvaccination protocol, which is limited by the number of metacercariaewhich can be used as challenge. By comparison animals which werevaccinated with adjuvant alone or control animals which received novaccine, were either dead or infected with at least one adult worm atthe end of the experiment (30 days after challenge infection). Of thelimited set of peptides selected for vaccination trials in F. hepatica,peptides 1.4 and 2.1 were the most effective, generating 100% protectionwith the protocol adopted, similar to that achieved with the wholeprotein. In this case peptides 1.1 and 1.3 were less effective (66.7%and 50% respectively) and peptide 2.5 was the least effective of all(12.5% protection).

In general terms these results are consistent with those for S. mansoni.However, in the case of the first family, there does seem to be somegain in using the designed fusion peptide 1.4 over the simpler peptide1.1. In this case, it would seem that there is also considerable gain inintroducing the glycines between the two original peptide segments,suggesting that additional flexibility may indeed help the peptide toassume an immunologically relevant conformation. In the case of family2, once again the larger peptide 2.5 was effectively inactive whilst thesmaller 2.1 (which is almost identical at its N-terminus) generated 100%protection.

Example 7 The Presence of FABPs related to Sm14 in Other Helminths ofMedical and Veterinary Importance

FIG. 14 shows the alignment of several FABPs from different parasites,highlighting elements of secondary structure and residues predicted toparticipate in the discontinuous epitopes of Sm14. These residues aretherefore potentially cross-reactive towards all parasites listed in thealignment.

In order to demonstrate the presence of cross-reactive molecules relatedto Sm14 in other helminthes, extracts from said helminthes were testedin the following manner.

Extracts from the following nine helmiths were tested: the trematodeEchinostoma paraensei; the cestoids Hymenolepis diminuta, Dipylidiumcaninum e Taenia saginata; the nematodes Aspiculuris tetraptera,Toxocara sp., Ascaris suum (machos), A. suum (fêmeas) e Toxocara canis.Four of these extracts (A. suum machos e fêmeas, E. paraensei e T.saginata) contained a protein component that was recognised bymonospecific polyclonal anti-rSm14 antibody, as demonstrated in FIG.16A. In the case of the extracts derived from both male and female A.suum, the cross-reacting component possesses a molecular weight of alittle over 14 kDa, as shown in FIG. 16B. The protein bands detected inthis experiment are extremely similar to those seen in the controls(saline extract, SE, from male and female parasites), in other words,they presented no differences in terms of expressibility as has beenseen in previous cases.

As previously stated FABPs have already been described in severaldifferent helminthes (including trematodes, cestoids and nematodes).Such molecules possess conserved amino acid sequences andthree-dimensional structures from one species to another. As an exampleSm14 and Fh15 (FABP de Fasciola hepatica) share 44% sequence identity,believed to be responsible for the heterologous resistance observedbetween the two species. A further example is the FABP from Ascarissuum, As-p18, which presents 28% sequence identity with Sm14 and a verysimilar predicted three-dimensional structure.

FIG. 16B also shows that a 14 kDa component from E paraensei isrecognized by anti-rSm14 serum, the latter obtained from rabbitspreviously inoculated with the recombinant molecule.

The final extract to show immune cross-reactivity with anti-rSm14 serumwas that from Taenia saginata. FIG. 16B also shows that in this case theprotein band recognized has a molecular weight of approximately 14 kDa.

These results demonstrate that the Sm14 molecule presents immunecross-reactivity with other proteins of the same family (FABPs from A.suum, E. paraensei e T. saginata.) Furthermore, in the case of infectionby F. hepatica, the efficiency of Sm14 as a vaccine against thefascioliasis has already been proven (see previous examples). Thisdemonstrates the effectiveness of the use of Sm14 as a bivalentanti-helminth vaccine for use against schistosomiasis and fascioliasis.

The fact that a) the above data demonstrate the phenomenon of immunecross-reactivity between anti-rSm14 serum and FABPs derived from severalother helminth parasites, b) FIG. 14 shows that many of the residuesidentified in Sm14 as belonging to the discontinuous epitopes areconserved in the sequences of FABPs from other parasitic helminthes, c)the fact that such molecules are well distributed amongst helminthparasites, d) the fact that the three-dimensional structure of suchFABPs is extremely well conserved, indicate that we can apply the Sm14molecule as the molecular basis for a multivalent anti-helminth vaccine.

The results of the Examples of the present invention show that uncoupledpeptides confer protection against both parasites which are equivalentto those seen with the parent molecule.”

The results presented in these examples also provide evidence that it ispossible to considerably simplify the Sm14 molecule and still induce aprotective immune response to both S. mansoni and F. hepatica inexperimental animals. This represents the development of a bivalentanti-helminth peptide vaccine, which provides several advantages interms of chemical stability, safety and reproducibility of manufacture,which have important consequences for efficient delivery in endemicregions.

In the case of humoral responses the three-dimensional conformationsaccessible to the peptide are relevant to their effectiveness and inprincipal, it is of interest to include as many of the regions of adiscontinuous epitope as possible into a single peptide.

Referring to FIG. 15, it represents the position of cysteine residues inpeptides and their role in restraining conformational movement. Residueswhich restrict the conformational mobility in relation to the elementsof the secondary structure were studied. Specifically, FIG. 15 shows theresult of the addition of cysteine as a tool for the restriction of thespatial mobility. It was found that the cysteines when present in pairin the sequence, for example in oxidant medium, form cistines(dissulphete bridge), joining peptide fragments which could be distantin the conformational structure in solution.

The result shows that the presence of FABPs in helminths is a rule,being the helminth a trematode, a cestoid or a nematode. In addition tothis, there exists the confirmation of the importance of FABPs as a basefor the vaccination, as they exhibit immunogenic epitopes and developimportant functions in the physiology of the microrganisms.

It should be noted that modifications and variations together withothers that would be obvious for a person of ordinary skill in the artare deemed to be within the scope of the present invention the nature ofwhich is to be determined from the above description and claims.

1. Method for constructing peptides on the basis of thethree-dimensional structures of homologous molecules wherein saidpeptides are able to mimic or prevent the interaction between helminthpathogens and receptors, said method consisting of: (i) selectingregions of the parent protein which contain a high spatial density ofresidues which form continuous or discontinuous epitopes; (ii) givepriority to maintaining the previously predicted epitopic residues,responsible for stimulating the desired immuno/antigenic response,within the selected peptide sequence; (iii) elaborate sequences which,whilst maintaining a high percentage of previously identified epitopicresidues, are of limited size varying from 8 to 28 residues; (iv) twopeptides may be chosen, which may correspond to regions which aredistant in the primary structure (amino acid sequence) but spatiallyclose in the tertiary (three-dimensional) structure, the said regionsare chosen on the basis that the three-dimensional structure indicatesthat they can be readily united assuming that they retain their originalstructures. (v) modifications may be made to the peptide, be it derivedfrom a single continuous stretch of amino acids or be it derived in themanner described in (iv), so as to favour the three-dimensionalconformation as seen in the original protein, such modifications mayinclude substitutions of amino acids as well as insertions or deletionsof amino acids; (vi) residues may be used in order to restrict theconformational freedom of the final peptide.
 2. Method according toclaim 1 wherein in step(vi) ½-cystines residues are used to restrict theconformational freedom of the final peptide.
 3. Method according toclaim 1 wherein the three-dimensional structure selected is thatcorresponding to a molecule of a Fatty Acid Binding Protein—FABP. 4.Method according to claim 1 wherein the three-dimensional structureselected is that of a molecule/protein from helminths, morepreferentially, Sm14 and homologous molecules.